Parenteral and oral formulations of benzimidazoles

ABSTRACT

Pharmaceutical compositions of a benzimidazole or a benzimidazole derivative are disclosed. For example, in certain embodiments the pharmaceutical compositions include a benzimidazole, PEG 400, and a dipolar aprotic solvent. In other embodiments, pharmaceutical compositions include a benzimidazole, an oil, a dipolar aproptic solvent, and a surfactant. In certain embodiments, the benzimidazole is mebendezole. The pharmaceutical compositions are formulated for delivery to a subject by any means, and include formulations for oral and parenteral delivery.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of pharmacology and pharmaceutics. More particularly, it concerns pharmaceutical compositions that include a benzimidazole, a polyol, and a dipolar aprotic solvent. It also concerns pharmaceutical compositions that include a benzimidazole, an oil, a dipolar aproptic solvent, and a surfactant.

2. Description of Related Art

The identification of improved methods of drug delivery is crucial in the treatment of a vast number of human diseases. In particular, even though scientific studies might have identified the potential usefulness of a certain drug in the treatment of a particular condition, treatment options may be limited if proper formulations of the drug to facilitate delivery of the agent to the diseased tissue are not identified.

One disease wherein pharmaceutics has played a significant role is cancer. Cancer is the second leading cause of death in the United States. Half of all men and one-third of all women in the United States will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is identified and treatment initiated, the greater are the chances for survival.

Pharmaceutical preparations that are effective against cancer comprise an emerging and expanding area of research and potential commercial development. Pharmaceutical agents are being developed that can delay or arrest development of cancer, and the development of precancerous lesions into cancers. Precancerous lesions include, for example, lesions of the breast that can develop into breast cancer, lesions of the skin that can develop into malignant melanoma or basal cell carcinoma, colonic adenomatous polyps that can develop into colon cancer, cervical dysplasia that can develop into cervical cancer, premalignant lesions of the oropharynx that can develop into head and neck cancer.

The search for drugs useful for treating and preventing neoplasias in their earliest stages is intensive because chemotherapy and surgery alone are often ineffective, and current cancer chemotherapy has severe side effects. Such preventive treatment is also potentially useful for recovered cancer patients who retain a risk of cancer recurrence, and even for cancer patients who would benefit from compounds that selectively induce apoptosis in neoplastic, but substantially not in normal cells.

Induction of apoptosis is one mechanism by which pharmaceutical agents can kill cancer cells. Apoptosis, sometimes referred to as “programmed cell death,” naturally occurs in many tissues in the body. It plays a critical role in tissue homeostasis, that is, it ensures that the number of new cells produced are correspondingly offset by an equal number of cells that die. Apoptosis is especially pronounced in self-renewing tissues such as bone marrow, immune cells, gut, and skin.

Standard chemotherapeutics can promote apoptosis not only in cancer cells, but also in normal human tissues. These agents often have particularly severe effect on tissues where apoptosis is especially pronounced (e.g., hair, gut, and skin). Thus, standard chemotherapeutics are inappropriate for cancer prevention, particularly if chronic administration is indicated.

Benzimidazoles (BZs) are a broad-spectrum class of antihelmintics that display excellent activity against parasitic nematodes and, to a lesser extent, against cestodes and trematodes. BZs have also been shown to be very effective antiprotozoal agents that also have antifungal activity. It is currently believed that BZs exert their cytotoxic effects by binding to the microtubule system and disrupting its functions (Lacey, 1988; Friedman and Platzer, 1980). The suggestions that tubulin is a target for BZs has been supported by the results of drug-binding studies using enriched extracts of helminth and mammalian tubulin (Lacey, 1988). Moreover, competitive drug-binding studies using mammalian tubulin have shown that BZs compete for colchicine binding and inhibit growth of L1210 murine leukemia cells in vitro (Friedman and Platzer, 1978; Lacey and Watson, 1989). However, BZs are selectively toxic to nematodes when administered as antihelmintics but are not toxic to the host. In contrast, BZs suppress the in vitro polymerization of mammalian tubulin. Differences in both the affinity between the host and parasite macromolecules for BZ (Russell et al., 1992; Kohler and Bachmann, 1981) and the pharmacokinetics of BZs between the host and the parasite have been suggested as responsible for the selective toxicity of BZs (Gottschall et al., 1990) but the actual molecular basis of this selective toxicity remains unclear.

Mebendazole (MZ), or 5-benzoyl-2-benzimidazole carbamic acid methyl ester, is a member of the BZ class of compounds. Recently, MZ has been found to induce mitotic arrest and apoptosis by depolymerizing tubulin in non-small cell lung cancer cells. (Sasaki et al., 2002). MZ has also been found to elicit a potent antitumor effect on human cancer cell lines both in vitro and in vivo (Mukhopadhyay et al., 2002).

MZ was first introduced for the treatment of roundworm infections as a result of research carried out by Brugmans et al. (1971). It is the prototype of a series of broad-spectrum anthelmintics widely used in both animals and man (Michiels et al., 1982) as broad-spectrum anthelmintics for animal and human use (Van den Bossche et al., 1982). Related BZ derivatives with anthelmintic properties include albendazole and flubendazole.

MZ is highly effective in ascariasis, intestinal capillariasis, enterobiasis, trichuriasis, and hookworm (Ancylostoma duodenale and Necator americanus) infection as single or mixed infections. The drug is active against both larval and adult stages of the nematodes that cause these infections, and it is ovicidal for Ascaris and Trichuris (Keystone and Murdoch, 1979; Van den Bossche et al., 1982). Immobilization and death of susceptible gastrointestinal organisms occurs slowly, and clearance from the gastrointestinal tract may not be complete until a few days after treatment with MZ. Together with albendazole, MZ has shown some promise in the treatment of hydatid disease (Wilson et al., 1987).

MZ causes selective disappearance of cyoplasmic microtubules in the tegumental and intestinal cells of affected worms. Secretory substances accumulate in Golgi areas, secretion of acetylcholinesterase and uptake of glucose are impaired, and glycogen is depleted. These effects of MZ are not noted in host cells. MZ has a high affinity for parasite tubulin in vitro, but it also binds to host tubulin. The biochemical basis for its selective action is thus unclear (see Van den Bossche, 1981; Watts et al., 1982).

The conventional formulation of MZ is a tablet form. Tablets of MZ are poorly and erratically absorbed, and concentrations of the drug in plasma are low and do not reflect the dosage taken (Witassek et al., 1981). This is because MZ is highly lipophilic, with an aqueous solubility of less than 1 μg/ml. As a result, the conventional formulations of MZ result in low bioavailability of the drug and erratic absorption from the gastrointestinal tract. Many other BZs and BZ derivatives are also highly lipophilic and erratically absorbed from the gastrointestinal tract.

Therefore, improved oral pharmaceutical compositions of BZs and BZ derivatives, such as MZ, that result in greater bioavailability of the drug, are needed to treat systemic diseases such as cancer and deep-seated parasitic diseases with extraintestinal manifestations. In addition, parenteral formulations that result in greater release of BZs and BZ derivatives compared to conventional formulations would also be beneficial in treating these systemic conditions. Pharmaceutical compositions such as these would result in greater concentration of the drug in the bloodstream, and consequentially greater efficacy and therapeutic benefit.

SUMMARY OF THE INVENTION

The present invention is based on the development of novel formulations BZ and BZ derivatives that result in improved drug solubility, improved drug release from the formulation, and improved bioavailability. Higher concentration of BZ or BZ derivative in the blood would result in greater therapeutic effect of this class of agents for use in the treatment of disorders in which treatment with BZs or BZ derivatives would be beneficial. For example, in the case of MZ, these diseases would include hyperproliferative diseases such as cancer, and extraintestinal and deep-seated parasitic disease such as hydatid disease.

The inventors have formulated MZ using co-solvency and microemulsion approaches which have yielded a drug concentration of 1.4-3.5 mg/ml, which is a 1,400-3,500 fold increase in solubility compared to conventional formulations of MZ. The inventors have assessed the formulations of MZ by in vitro USP dissolution, in vivo bioavailability studies in rats, and cell growth inhibition in cell line cultures of numerous types of cancer cells. A substantial improvement in release of the MZ from the formulations was demonstrated compared to an unformulated suspension, and bioavailability was improved 130 fold. An enhanced IC₅₀ of MZ from the formulations for cell growth in lung cancer cell lines was demonstrated compared to control formulations dissolved in dimethylsulfoxide and the formulations exhibited favorable characteristics in preclinical pharmacokinetic and pharmacodynamic studies. Therefore, the new BZ formulations are expected to have enhanced efficacy in the treatment of cancer and other hyperproliferative diseases.

Certain embodiments of the present invention are pharmaceutical compositions that include a BZ, a polyol, and a dipolar aprotic solvent. The definition of pharmaceutical composition is discussed in detail elsewhere in this specification, and encompasses a composition that does not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate.

Any BZ, BZ derivative, or combination of BZs is contemplated for use in the pharmaceutical compositions of the present invention. One of skill in the art would be familiar with this broad class of agents, and would understand that the pharmaceutical compositions of the present invention pertain to use of any member or members of this class of drugs. For example, the BZ included in the present pharmaceutical compositions may be a derivative having the formula:

wherein R³ is selected from the group that includes H, carboxyl (—CO₂H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, methoxy(methoxy-dimethyl, pyridinyl)methyl-(sulfonyl), fluorophenylmethyl-2-chloro, propenyl, chloropropyl or esters (—CO₂R⁴) wherein R⁴ is selected from the group that includes alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH₃CH₂(OCH₂CH₂)n-, or CH₃CH₂CH₂(OCH₂CH₂CH₂)n-, or (CH₃)₂CH(OCH(CH₃)CH₂)n-, wherein n is from 1-3, wherein R¹ is OH, Cl, SH, carbamate or piperidin-4-yl, and R² is hydrogen, α-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof.

In certain embodiments of the present invention, the BZ derivative is methyl 5-benzoylbenzimidazole-2-carbamate (mebendazole, MZ). Alternatively, in other embodiments, the BZ derivative may be methyl 5-(phenylthio)-2-benzimidazole carbamate (fenbendazole) or 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole (omeprazole).

In certain other embodiments, the BZ derivative in the pharmaceutical compositions may be

Alternatively, the BZ derivative of the pharmaceutical compositions may be

In still other embodiments, the pharmaceutical composition includes a BZ derivative that is

The pharmaceutical compositions of the present invention contemplate use of any polyol. One of ordinary skill in the art would be familiar with the class of agents known as polyols, and would understand that any of these agents may be included in the pharmaceutical compositions of the present invention. For example, the polyol may be polyethylene glycol (PEG) 200, PEG 300, PEG 400, PEG 600, 1,2-propylene diol, glycerol, or ethylene glycol. In certain embodiments, the polyol may be PEG 400. The pharmaceutical compositions of the present invention can include a single polyol, or more than one polyol.

The pharmaceutical compositions of the present invention include dipolar aprotic solvents. A dipolar aprotic solvent is a solvent with a comparatively high relative dielectric constant, greater than about 15, and a sizable permanent dipole moment, that cannot donate suitably labile hydrogen atoms to form strong hydrogen bonds. The term is something of a misnomer, since such solvents are usually not aprotic but protophilic (and at most, weakly protogenic). One of ordinary skill in the art would be familiar with the class of agents typically known as dipolar aprotic solvents, and would understand that any of these agents may be included in the pharmaceutical compositions of the present invention.

For example, the dipolar aprotic solvents include N,N-dimethylacetamide (DMA) or dimethylsulfoxide (DMSO). In certain embodiments, the pharmaceutical compositions include N,N-dimethylacetamide as the dipolar aprotic solvent. In certain other embodiments, the dipolar aprotic solvent is dimethylsulfoxide. In some embodiments, the pharmaceutical compositions include more than one dipolar aprotic solvent. For example, the pharmaceutical compositions may include N,N-dimethylacetamide and dimethylsulfoxide.

The pharmaceutical compositions of the present invention contemplate any ratio of polyol to dipolar aprotic solvent by weight. However, in certain embodiments of the present invention, the ratio of polyol to dipolar aprotic solvent by weight is 3:1. For example, the pharmaceutical compositions of the present invention may include PEG 400 and N,N-dimethylacetamide in a ratio of 3:1 by weight. Alternatively, certain embodiments of the present invention may include PEG 400 and dimethylsulfoxide in a 3:1 ratio by weight.

In some embodiments of the present pharmaceutical compositions, the ratio of polyol to dipolar aprotic solvent is 7:1 by weight. For example, the pharmaceutical may include PEG 400 and dimethylsulfoxide in a ratio of 7:1 by weight, or PEG 400 and N,N-dimethylacetamide in a ratio of 7:1 by weight.

In certain embodiments of the present pharmaceutical compositions, the composition includes water. Any amount of water is contemplated by the present invention. However, in certain embodiments, the relative ratio of polyol:dipolar aprotic solvent:water is 3:1:1 by weight. For example, some embodiments include a pharmaceutical compositions that may include PEG 400:N,N-dimethylacetamide:water in a 3:1:1 ratio by weight. Alternatively, other embodiments of the present pharmaceutical compositions include PEG 400:dimethylsulfoxide:water in a 3:1:1 ratio by weight.

In other embodiments of the present pharmaceutical compositions, the relative ratio of polyol:dipolar aprotic solvent:water is 7:1:2. For example, the pharmaceutical composition may include PEG 400:dimethylsulfoxide:water in a 7:1:2 ratio by weight.

Additional embodiments of the present invention pertain to pharmaceutical compositions that include (1) a BZ; (2) an oil; (3) a dipolar aprotic solvent; and (4) a surfactant. Members of the class of BZ and BZ derivatives are discussed above, and elsewhere in this specification. As discussed above, one of skill in the art would be familiar with this broad class of agents, and would understand that the pharmaceutical compositions of the present invention pertain to use of any member or combination of members of this class of drugs. For example, as discussed above, the BZ derivative may be MZ.

Any oil is contemplated for use in the present invention. One of ordinary skill in the art would be familiar with the wide variety of oils that are available for inclusion in pharmaceutical compositions. For example, the oil may be super refined corn oil, super refined cottonseed oil, olive oil, super refined peanut oil, super refined soybean oil, Captex 200, Captex 355, Miglyol 812, or Myvacet 9-45. In certain embodiments of the present pharmaceutical compositions, the oil is Captex 200. In certain other embodiments of the present pharmaceutical compositions, the oil is Myglyol 812. The pharmaceutical compositions of the present invention also contemplate compositions containing more than one oil or any combination of oils.

Any surfactant is contemplated for use in the present invention. One of ordinary skill in the art would be familiar with the wide variety of surfactants that are available for inclusion in pharmaceutical compositions of the present invention. For example, in certain embodiments, the pharmaceutical compositions of the present invention include Tween 80 as the surfactant. In other embodiments, the pharmaceutical compositions include Arlacel 80 as the surfactant. The pharmaceutical compositions of the present invention may include a single surfactant, or any combination of surfactants.

Surfactants of any hydrophilic-lipophilic balance (HLB) are contemplated for use in the present invention. The HLB of a surfactant is an empirical quantity, on an arbitrary scale, which is a measure of the polarity of a surfactant or mixture of surfactants. It is a widely known and used term which would be very familiar to one of ordinary skill in the art. In general, a high HLB surfactant is a surfactant with an HLB that is 7 or above 7, and a low HLB surfactant is a surfactant with an HLB below 7. More than one surfactant in the composition may provide for versatility, greater stability, and enhanced drug absorption. For example, some embodiments of the present invention include a combination of Tween 80 and Arlacel 80 as the surfactants.

In certain embodiments of the present pharmaceutical compositions, the surfactants used are only high HLB surfactants. Other embodiments of the present pharmaceutical compositions include only low HLB surfactants. Alternatively, the pharmaceutical compositions of the present invention include a mixture of both high HLB surfactants and low HLB surfactants.

As discussed above, the pharmaceutical compositions of the present invention may further be defined as including water. Any amount of water is contemplated in the present pharmaceutical compositions, as long as any amount of the previously defined components is present.

The pharmaceutical compositions of the present invention may further be defined as a microemulsion. A microemulsion is herein defined as a thermodynamically stable dispersion of one liquid phase into another, stabilized by an interfacial film of surfactant. This dispersion may be either oil-in-water or water-in-oil. Microemulsions are often clear solutions, as the droplet diameter may be approximately 100 nanometers or less. However, solutions that are not clear are encompassed within the definition of microemulsion used herein, and any droplet diameter is contemplated in this definition.

Examples of certain embodiments of the present invention include pharmaceutical compositions that include (1) MZ; (2) 10%-60% by weight of an oil; (3) 2%-30% by weight of a dipolar aprotic solvent; (4) 5%-50% by weight of a first surfactant; and (5) 5%-50% by weight of a second surfactant.

The pharmaceutical compositions of the present invention contemplate any concentration of MZ. For example, in certain embodiments the concentration of MZ is greater than 1.4 mg/ml. For example, the MZ concentration may be about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg.ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about 3.1 mg/ml, about 3.2 mg/ml, about 3.3 mg/ml, about 3.4 mg/ml, about 3.5 mg/ml, or any range of concentration or increments of concentration derivable therein.

In other embodiments, the concentration of MZ is 0.3-3.0 mg/ml. For example, the concentration of MZ may be about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, or any range of concentration or increment of concentration derivable therein. In other embodiments, the concentration of MZ is 0.5 mg/ml to 2.5 mg/ml or 0.3 mg/ml to 3.0 mg/ml.

In certain specific embodiments, the concentration of MZ is about 1.2 mg/ml. In other embodiments, the concentration of MZ is about 1.5 mg/ml. In still other embodiments, the concentration of MZ is about 1.7 mg/ml. In additional embodiments, the MZ concentration is about 1.8 mg/ml. In other embodiments, the MZ concentration is about 1.85 mg/ml. In other embodiments, the MZ concentration is about 1.9 mg/ml. In further embodiments, the MZ concentration is about 2.0 mg/ml. In still further embodiments, the MZ concentration is about 2.1 mg/ml. In certain other embodiments, the concentration of MZ is about 2.3 mg/ml. In further embodiments, the MZ concentration is about 2.6 mg/ml.

The concentration of BZ in the pharmaceutical compositions of the present invention may be determined by any method known to those of skill in the art. One of ordinary skill in the art would be familiar with the range of techniques used to determine concentration of a BZ or BZ derivative in a composition. For example, the concentration of BZ may be determined by HPLC. In certain embodiments wherein the BZ derivative is MZ, HPLC may be used to determine the concentration of MZ. Use of HPLC to determine concentration of MZ in a pharmaceutical composition of the present invention is discussed further in the specification below.

In some embodiments of the present invention, the pharmaceutical compositions may include water. Any amount of water is contemplated in the present invention, as long as the other required constituents are present. In addition, the pharmaceutical compositions of the present invention contemplate the presence of components in addition to those that have been delineated. A greater discussion of potential additional additives is discussed in the specification below.

Some embodiments of the present pharmaceutical compositions include Tween 80 as the first surfactant and Arlacel 80 as the second surfactant. For example, in some embodiments, the pharmaceutical compositions include 10%-60% by weight of Captex 200, 3%-30% by weight of N,N-dimethylacetamide, 5%-50% by weight of Tween 80, 5%-50% by weight of Arlacel 80, and 2%-20% by weight of water. In other embodiments, the pharmaceutical composition includes 28.9% by weight of Captex 200, 13.7% by weight of N,N-dimethylacetamide, 28.8% by weight of Tween 80, 28.8% by weight of Arlacel 80, and 13% water.

In certain other embodiments, the pharmaceutical composition includes 10%-60% by weight of Captex 200, 2%-20% by weight of dimethylsulfoxide, 10%-40% by weight of Tween 80, 10%-40% by weight of Arlacel 80, and 10%-20% by weight of water. For example, the pharmaceutical composition may include 50.1% by weight of Captex 200, 12.6% by weight of dimethylsulfoxide, 12.6% by weight of Tween 80, 12.6% by weight of Arlacel 80, and 12.1% by weight of water. The pharmaceutical composition may also include 42.6% by weight of Captex 200, 10.7% by weight of dimethylsulfoxide, 16.1% by weight of Tween 80, 16.1% by weight of Arlacel 80, and 14.5% by weight of water. In other examples, the pharmaceutical compositions includes 38.4% by weight of Captex 200, 9.6% by weight of dimethylsulfoxide, 19.2% by weight of Tween 80, 19.2% by weight of Arlacel 80, and 13.6% by weight of water. In further examples, the pharmaceutical composition includes 34.7% by weight of Captex 200, 8.7% by weight of dimethylsulfoxide, 21.7% by weight of Tween 80, 21.7% by weight of Arlacel 80, and 13.2% by weight of water.

The pharmaceutical compositions of the present invention may also include 10%-60% by weight of super refined soybean oil, 2%-20% by weight of dimethylsulfoxide, 10%-40% Tween 80, 10%-40% Arlacel 80, and 0.5%-15% by weight of water. For example, the pharmaceutical composition may include 56.5% by weight of super refined soybean oil, 14.1% by weight of dimethylsulfoxide, 14.1% by weight of Tween 80, 14.1% by weight of Arlacel 80, and 1.2% by weight of water. In other embodiments of the present invention, the pharmaceutical composition includes 44.2% by weight of super refined soybean oil, 11.1% by weight of dimethylsulfoxide, 16.6% by weight of Tween 80, 16.65 by weight of Arlacel 80, and 11.5% by weight of water. In further embodiments, the pharmaceutical composition includes 40.0% by weight of super refined soybean oil, 10.0% by weight of dimethylsulfoxide, 20.0% by weight of Tween 80, 20.0% by weight of Arlacel 80, and 10.0% by weight of water. In additional embodiments, the pharmaceutical composition includes 35.7% by weight of super refined soybean oil, 8.9% by weight of dimethylsulfoxide, 22.3% by weight of Tween 80, 22.3% by weight of Arlacel 80, and 10.8% by weight of water.

Embodiments of the present invention also include pharmaceutical compositions that include 10%-60% by weight of Miglyol 812, 2%-20% by weight of dimethylsulfoxide, 10%-40% by weight of Tween 80, 10%-40 by weight of Arlacel 80, and 5%-20% by weight of water. For example, the pharmaceutical composition may include 50.4% by weight of Miglyol 812, 12.6% by weight of dimethylsulfoxide, 12.7% by weight of Tween 80, 12.7% by weight of Arlacel 80 and 11.6% by weight of water. In other examples, the pharmaceutical composition includes 42.4% by weight of Miglyol 812, 10.6% by weight of dimethylsulfoxide, 15.9% by weight of Tween 80, 15.9% by weight of Arlacel 80, and 15.2% by weight of water. In other examples, the pharmaceutical compositions include 39.2% by weight of Miglyol 812, 9.8% by weight of dimethylsulfoxide, 19.6% by weight of Tween 80, 19.6% by weight of Arlacel 80, and 11.8% by weight of water. The pharmaceutical composition of the present invention may also include 34.7% by weight of Miglyol 812, 8.7% by weight of dimethylsulfoxide 21.8% by weight of Tween 80, 21.8% by weight of Arlacel 80, and 13.0% by weight of water.

The present pharmaceutical compositions can be formulated by any method known to those of skill in the art. For example, in certain embodiments the pharmaceutical composition is formulated for parenteral administration to a subject. Parenteral administration is defined as administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

In other embodiments of the present invention, the pharmaceutical composition is further defined as being suitable for administration through the digestive tract or through non-invasive topical and regional routes. For example, the pharmaceutical preparation may be administered orally. In other embodiments, the pharmaceutical preparation is administered intranasally, topically, locally, by inhalation, by continuous infusion, or by localized perfusion by bathing target cells directly, via a catheter, or via lavage.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 Authentic HPLC chromatogram of MZ (0.1 μg/mL, 100 μL=10 ng).

FIG. 2 Representative pseudo-ternary phase diagram of the microemulsion formulations described in Table III-E.

FIG. 3 In vitro release of MZ from microemulsion of formula codes A4 and E4.

FIG. 4 Mean pharmacokinetic profile (n=3) of MZ after intravenous bolus administration of co-solvency formula P7 (3.25 mg/kg) to rats.

FIG. 5 Mean pharmacokinetic profile (n=4) of MZ after oral administration of microemulsion E4 (4.89 mg/kg) to rats.

FIG. 6 Mean pharmacokinetic profile (n=4) of MZ after oral administration of suspension formulation (50 mg/kg) to rats.

FIG. 7 Semi-logarithmic pharmacokinetic profiles of MZ after intravenous bolus administration of co-solvency formulation P7 (3.25 mg/kg) to rats.

FIG. 8. Cytotoxicity of MZ E4-microemulsion in three skin cancer cell lines (SRB1, A375, 2237).

FIG. 9. Cell killing effects of MZ E4-microemulsion and MZ-DMSO versus placebos in H1299 cells.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention seeks to exploit the inventors' discovery by providing pharmaceutical compositions of BZs and BZ derivatives that have improved and predictable bioavailabilities. BZs and BZ derivatives often have limited and erratic oral bioavailability due to poor solubility of the compound in aqueous medium. As a result, these drugs cannot be efficiently delivered in convention dosage forms. For example, MZ has a solubility in aqueous medium of less that 1 μg/ml. The inventors have discovered certain oral and parenteral formulations of BZs and BZ derivatives, such as MZ, using co-solvency and microemulsion approaches, respectively. The parenteral formulation of MZ was developed to circumvent the drawback of extensive first-pass metabolism of MZ known to be associated with oral administration. These formulations can be applied for the therapy of diseases wherein BZs and BZ derivatives formulated for increased bioavailability may be efficacious, such as hyperproliferative diseases, neoplastic disease and extraintestinal parasitic diseases.

A. Benzimidazoles and Derivatives of Benzimidazoles

Benzimidazoles (BZs) are broad-spectrum antihelmintics that display excellent activity against parasitic nematodes and, to a lesser extent, against cestodes and trematodes. BZs have also been shown to be very effective antiprotozoal agents and to have antifungal activity. It is currently believed that BZs exert their cytotoxic effects by binding to the microtubule system and disrupting its functions (Lacey, 1988; Friedman and Platzer, 1980). The suggestion that tubulin is a target for BZs has been supported by the results of drug-binding studies using enriched extracts of helminth and mammalian tubulin (Lacey, 1988). Moreover, competitive drug-binding studies using mammalian tubulin have shown that BZs compete for colchicine binding and inhibit growth of L1210 murine leukemia cells in vitro (Friedman and Platzer, 1978; Lacey and Watson, 1989). However, BZs are selectively toxic to nematodes when administered as antihelmintics but are not toxic to the host. In contrast, BZs suppress the in vitro polymerization of mammalian tubulin. Differences in both the affinity between the host and parasite macromolecules for BZ (Russell et al., 1992; Kohler and Bachmann, 1981) and the pharmacokinetics of BZs between the host and the parasite have been suggested as responsible for the selective toxicity of BZs (Gottschall et al., 1990) but the actual molecular basis of this selective toxicity remains unclear.

Benzimidazoles and derivatives of benzimidazoles are defined herein to have the formula:

wherein R³ is selected from the group consisting of H, carboxyl (—CO₂H), hydroxyl, amino or esters (—CO₂R′) wherein R′ is selected from the group consisting of is selected from the group that includes alkoxy, haloalkyl, alkenyl, and cycloalkyl wherein the alkyl groups have from 1-8 carbons or CH₃CH₂(OCH₂CH₂)n-, or CH₃CH₂CH₂(OCH₂CH₂C₂)n- or (CH₃)₂CH(OCH(CH₃)CH₂)n- wherein n is from 1-3 and the pharmaceutically acceptable organic or inorganic addition salts thereof; wherein R³ is selected from the group consisting of H, carboxyl (—CO₂H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, methoxy(methoxy-dimethyl, pyridinyl)methyl-(sulfonyl), fluorophenylmethyl-2-chloro, propenyl, chlorophyl or esters (—CO₂R⁴) wherein R⁴ is selected from the group consisting of alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH₃CH₂(OCH₂CH₂)n-, or CH₃CH₂CH₂(OCH₂CH₂CH₂)n-, or (CH₃)₂CH(OCH(CH₃)CH₂)n-, wherein n is from 1-3, wherein R¹ is OH, Cl, SH, carbamate or piperidin-4-yl, and R² is hydrogen, α-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof. For R⁴ the preferred alkyl groups are straight chain, the preferred halogen is substituted on the terminal carbon, the preferred halogen is chlorine, the preferred cycloalkyl groups are those having 3-6 carbon atoms, and the cycloalkyl groups also include those which are substituted on an alkyl chain, 2-cyclopropylethy, cyclopropylmethy, 2-cyclopropylpropyl or 2-cyclopropylpropyl or cyclohexylmethyl.

BZs have some anti-tumor growth properties in vitro (WO 98/513304; WO 98/32440). The efficacy of BZs in vivo as an anti-tumor treatment has been limited to tumors already in regression following chemotherapeutic treatment.

Alternative benzimidazoles are: fenbendazole, albendazole, albendazole sulfone, oxibendazole, rycobendazole, thiabendazole, oxfendazole, flubendazole and carbendazim. Alternative anti-helminthic drugs are the imidazoles: niridazole and levimasole, the piperazines: piperazine and diethylcarbamazine, the isothiocyanates: amoscanate and CGP 6140. In addition, suramin, ivermectin, hycanthone, metrifonate, oxamniquine and praziquantel are anti-helminthic drugs.

B. Treatment of Cancers Using Benzimidazoles

There is evidence, as discussed above, that BZs or BZ derivatives may be useful in the treatment of hyperproliferative diseases, such as cancer. In particular, the use of BZs in the treatment of cancer is discussed in detail in U.S. patent application Ser. No. 10/043,877, which is specifically incorporated by reference herein. In addition, MZ has also been found to elicit a potent antitumor effect on human cancer cell lines both in vitro and in vivo (Mukhopadhyay et al., 2002; Liang et al., 2002). Mukhopadhyay et al., 2002, and Liang et al., 2002, are specifically incorporated by reference herein.

Of particular interest are patients that have wild-type tumor suppressor (e.g., p53) function. The tumor suppressor status of the tumor cells can be determined using any conventional methods, examples of which are described below. Patients may, but need not, have received previous chemo-, radio-, or gene therapies. Optimally, patients will have adequate bone marrow function (defined as peripheral absolute granulocyte count of >2000/mm³ and platelet count of 100,000/mm³), adequate liver function (bilirubin ≦1.5 mg/dl) and adequate renal function (creatinine <1.5 mg/dl).

Patients with cancer may be treated with a pharmaceutically acceptable form of BZ or a functional analog thereof. This administration could be in the form of, for example, an intratumoral injection, or indeed, any other method of application that is routinely used and well known to one of skill in the art, e.g., oral, systemic, local, regional. A biopsy of the lesions to be injected may be performed and the tissue stored for immunohistochemistry analyses.

The dose of BZ typically will be reconstituted into a pharmaceutically acceptable form immediately prior to administration. The dose of BZ will be determined depending on the clinical condition to be treated. For example, if the disease is cancer, the starting dose will be approximately 0.1 to 1 mg BZ /kg body weight. Of course, this may vary depending on the disease to be treated, the size of the tumor, the rate at which the tumor is growing, etc.

C. Pharmaceutical Compositions and Routes of Administration

1. Overview

The objective of the present invention was to develop both parenteral and oral formulations of BZ and BZ derivatives, such as MZ, in order to achieve improved and predictable bioavailabilities. The invention stems from the desire to overcome the problem of limited and erratic oral bioavailability due to poor solubility in aqueous medium of many BZs and BZ derivatives. The parenteral and oral formulations of BZs described in greater detail below were developed using co-solvency and self-emulsifying/microemulsion approaches, respectively. The parenteral formulation of BZ were developed to circumvent the drawback of the extensive first-pass metabolism of MZ and other BZs known to be associated with oral administration.

The inventors have investigated the solubility of MZ in various solvents, such as N,N-dimethylacetamide (DMA), DMSO, and polyethylene glycol-400 (PEG), as discussed in greater detail in the examples below. As primary solvents, the solvents would be miscible in secondary solvents, examples of which are normal saline, dextrose in water (5% or 10%), and water. These solvents are examples of vehicles in which BZs such as MZ could be suitably solubilized, yet be safe for human administration, alone or in combinations with other drugs. The solubility of BZ in individual solvent vehicles is shown in Table I below.

Virtually no MZ is absorbed through the intestinal tract after oral administration, making it impossible to even investigate its use as an oral antimicrobial against systemic infections. Parenteral administration would therefore be the logical approach to evaluate MZ and other BZs as therapy for deep-seated, systemic fungal infections and also in the treatment of cancer and other hyperproliferative diseases.

2. Effective Amount of BZs and BZ Derivatives

Pharmaceutical compositions of the present invention will include an effective amount of a BZ or a BZ derivative or a mixture thereof that is clinically determined to be useful in the treatment of the particular disease under consideration. For example, an effective amount of a BZ or a BZ derivative in a patient with cancer may be an amount that promotes expression of wild-type tumor suppressor genes, and/or inhibits angiogenesis. Alternatively, an effective amount of a BZ or BZ derivative or mixture thereof may be an amount that promotes death of parasites and/or regression of clinical findings associated with the infection. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier.

In cancer patients, the access to parenteral BZs such as MZ will be particularly important, since their intestinal absorption is often perturbed after chemotherapy, aggravating the already erratic intestinal absorption of various medications. The parenteral route will also make it possible to circumvent unpredictable first-pass metabolic effects in the liver, well known to alter the bioavailability of numerous pharmacologically active agents after oral dosing. Further, the availability of MZ for effective and reliable systemic administration will for the first time make it possible to clinically compare the activity of MZ against other treatments for cancer and deep-seated fungal infections.

3. Definition of “Pharmaceutical”

The phrases “pharmaceutical,” “pharmaceutically,” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate. As used herein, “pharmaceutical” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents or anthelmintics, can also be incorporated into the compositions.

4. Oral Administration

In addition to the compounds formulated for parenteral administration, such as those for intravenous, subcutaneous, or intramuscular injection, other pharmaceutically acceptable forms include, solutions suitable for oral administration. The term “oral administration” includes any form of administration of drug through the oral cavity and into the gastrointestinal tract. The pharmaceutical compositions of the present invention may be formulated for delivery to a subject by any means. For example, the compositions may be formulated as mouthwashes, mouthrinses, solutions for administration through a nasogastric tube, and the like.

5. Compositions Suitable for Parenteral Administration

As discussed in the summary of the invention, the active compounds of the present invention will often be formulated for parenteral administration, e.g., formulated for injection by any route other than through the digestive tract, such as via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. The definition of parenteral administration is discussed further in the summary of the invention. Typically, the compositions can be prepared as injectables, either as liquid solutions or suspensions for any parenteral but intravenous routes; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

6. Formulation Principles

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitable for mixture with a surfactant, such as hydroxypropylcellulose or polysorbate. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions, and formulations including sesame oil, peanut oil or aqueous propylene glycol or other solvent as discussed in the examples below. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be chemically and physically stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Preparation of cosolvency formulations and microemulsion formulations of BZs or BZ derivatives is discussed in greater detail in the examples below.

The proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

In certain cases, the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in gels, creams, and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.

For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary. These particular solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

7. Formulation of BZs and BZ Derivatives that are Sparingly Soluble in Water

a. Co-solvency Systems

Many BZs are only sparingly soluble in aqueous solutions. As discussed above, it is therefore an aspect of the current invention that the BZ compound be in a formulation which allows for an increased solubilization of the BZ or for a more effective dispersion.

Generation of some of the pharmaceutical compositions of the present invention are based on the principle of cosolvency (Spiegel and Noseworthy, 1963; Yalkowsky and Roseman, 1981). The examples show that pharmaceutical compositions of MZ can be generated without destroying its anti-cancer properties. Further, the animal studies provide evidence that the preferred vehicles are nontoxic and safe for administration and should be acceptable for human administration in the proposed concentrations and total doses to be utilized; indeed, DMA, DMSO, and PG have been used for solubilization of various pharmacologically active agents used in man (NIH Pub. 84-2141, 1984; Weiss et al., 1962; Kim, 1988). The parenteral administration of PEG has been studied in detail in a simian model (Lockard et al., 1979), and PEG has subsequently been used clinically as a (covalently bound) carrier of L-asparaginase in the treatment of lymphocytic leukemia and lymphoma (Keating et al., 1993). DMSO is also extensively used as a cryoprotective agent for low-temperature storage of human bone marrow and peripheral blood derived hematopoietic stem cell preparations to be used for transplantation after high-dose chemotherapy (McGann, 1978; Gorin, 1986; Davis and Rowley, 1990; Gorin, 1992). No serious adverse effects have been experienced from the use of these vehicles. The clinical use of normal saline, dextrose in water (5-70%), and aqueous lipid emulsion are well established means to alter the fluid and electrolyte balance and to supply parenteral nutrition. Normal saline and dextrose in water are extensively used to dilute various medications for parenteral use.

b. Microemulsion Systems

The preparation and use of microemulsions in the formulation of drugs, proteins, and the like are known in the art. A microemulsion is herein defined as a thermodynamically stable dispersion of one liquid phase into another, stabilized by an interfacial film of surfactant. This dispersion may be either oil-in-water or water-in-oil. Microemulsions are often clear solutions, as the droplet diameter may be approximately 100 nanometers or less. However, solutions that are not clear are encompassed within the definition of microemulsion used herein, and any droplet diameter is contemplated in this definition.

Surfactants can be added to the aqueous solution to increase solubility and stability of a solution or dispersion. Surfactants, discussed in greater detail in the summary of the invention, are organic molecules which contain both hydrophilic and hydrophobic ends. The hydrophilic end, which is either polar or ionic, dissolves readily in water. The hydrophobic, or non-polar, end, however, does not dissolve in water and will move as far away from water as possible. When a small concentration of surfactant is added to water, the hydrophobic end will immediately rise to the surface or orient towards a less polar group. The addition of surfactants in a BZ solution increase the solubility and/or stability of the BZ-surfactant solution relative to the BZ alone.

D. Methods of Measuring Concentration of Drugs in a Composition

Following preparation of the pharmaceutical compositions of the present invention, it may be desirable to quantify the amount of BZ or BZ derivative in the pharmaceutical composition. Methods of measuring concentration of a drug in a composition include numerous techniques that are well-known to those of skill in the art. Selected examples include chromatographic techniques. There are many kinds of chromatography which may be used in the present invention: drug-specific assays, adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer chromatography, gas chromatography, and high performance liquid chromatography (HPLC). One of ordinary skill in the art would be familiar with these and other related techniques.

E. EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 Materials and Methods

Materials

Mebendazole (MZ, Lot 90K1145), polyethylene glycol (PEG) 400 (Lot 121H0052), N,N-demethylacetamide (DMA) (Lot 129H3498), and dimethylsulfoxide (DMSO) (Lot 94H0358) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Propylene glycol (Lot 940629) and olive oil (Lot CAS8001-25-0) were obtained from Wood Scientific, Inc (Houston, Tex.). Triacetin was purchased from Lancaster Synthesis, Inc. (Windham, N.H.). Super refined cottonseed oil (Lot GAB-217NP), super refined peanut oil (Lot BSS-429NP) and super refined soybean oil (Lot SB4-393NP) were supplied by Croda (Mill Hall, Pa). Captex 200 (C8/C10 diesters of propylene glycol, Lot 10505-6) and Captex 355 (C8/C10 triglycerides from coconut oil) were provided by Abitec Corporation (Janesville, Wisc.). Miglyol 812 (C8/C10 triglycerides from coconut oil, Lot 1995050866) and Myvacet 9-45 (distilled acetylated monoglycerides; Lot 960225) were obtained from Eastman Chemical Products. Tween 80 (polyoxytheylene [20] sorbitan monooleate; Lot 32332) and Arlacel 80 (sorbitan monooleate NF, NLB=43, Lot 27255) were supplied by ICI Americans Inc. (Wilmington, Del.). Cremophor RH 40 (PEG-40 hydrogenated castor oil, HLB=13.5) was from BASF (Parsippany, N.J.).

HPLC Assay of MZ

An HPLC assay was developed to quantify MZ in the formulations and in the release samples. The assay used a reversed-phase μBondapak C₁₈ column (300×3.9 mm, 10 μm, Waters Corporation, Milford, Mass.) preceded by a C₁₈ guard column. The mobile phase, which was filtered and degassed prior to use, consisted of acetonitrile/0.05 M potassium monobasic phosphate, pH 6.5 (40:60, v/v). Prednisone was used as the internal standard (IS). The flow rate was 1.2 ml/min. The solvent delivery system was a ConstaMetric 3500 LDC Analytical Model system. MZ and IS were detected with a SpectroMonitor 3200 programmable multiwavelength UV detector set at 254 nm. Peak area ratios measured by a Milton Ray CI-4100 data module integrator (LDC Analytical) were used to construct standard curves and to determine the concentrations of MZ in the samples. The assay was later modified for the quantification of MZ in blood samples, as described below.

Evaluation of MZ Solubility in Various Solvent Media

The solubility of MZ in aqueous and in fourteen (14) other solvent media, including six (6) pharmaceutical solvents (methanol, PEG 400, propylene glycol, triacetin, DMA, and DMSO) and eight (8) naturally occurring and synthetic oils (super refined cottonseed oil, olive oil, super refined peanut oil, super refined soybean oil, Captex 200, Captex 355, Myglyol 812, and Myvacet 9-45), were evaluated. The solubility of MZ in each solvent medium was determined by adding excessive amount of MZ to the medium, followed by shaking at room temperature for at least 48 hours. After the equilibrium was reached, an aliquot was withdrawn and filtered through a membrane with a pore size of 0.45 μm. The clear filtrate was diluted with an appropriate volume of DMA and analyzed by the developed HPLC assay.

Development of Parenteral Formulations Using Co-solvency Approach

One hundred milligrams of MZ were dissolved in 11 ml of DMA by warming in a 60° water bath. The solution was then mixed with PEG 400 at ratios of 1:1 to 1:7 (v/v). A final portion of water was added to obtain a final co-solvent formulation with volume ratios of DMA:PEG 400:water at 2:2:1, 1:3:1, and 1:7:2.

Using this approach, ten co-solvent systems of various compositions and ratios were evaluated and optimized to achieve the maximal drug solubility for parenteral delivery. The MZ solubility in individual co-solvent formulations was determined by the HPLC assay. The optimal formulation was selected to be administered intravenously (I.V.) as the most bioavailable formulation reference in the preclinical bioavailability study of the selected lead oral microemulsion formulation.

Development of Microemulsion Formulations

Six self-emulsifying drug delivery systems (SEDDS) were prepared by mixing an oil, a low HLB surfactant, and a high HLB surfactant. In particular, 100 mg of MZ were dissolved in 5.5 ml of DMSO or DMA at 60 degree C. in a water bath. The solution was then mixed with Tween 80, Arlacel 80, and various types of oils at different ratios.

A clear, transparent formulation was indicative of the formation of a stable microemulsion. Phase diagrams were constructed with six different ratios of the ingredients for each system, by adding a small increment of an aqueous phase (deionized water) to the SEDDS until the system became turbid, reflecting a phase separation, to define the boundary of the composition region for stable microemulsions.

Once the region of stable microemulsions was identified on the phase diagram, microemulsions were prepared by mixing appropriate quantities of the four ingredients (oil, dipolar aprotic solvent, surfactant, and water) with a gentle hand shaking or stirring to ensure a through mixing. MZ, either dissolved in DMA or DMSO, was incorporated by mixing with the other ingredients.

Two optimal microemulsion formulations were selected for further assessment of in vitro drug release. One of the two optimal formulations was evaluated for cell growth inhibition in cell culture models, and for in vivo preclinical bioavailability in rats. The lead microemulsion formulation will be suitable for future evaluation of anti-neoplastic efficacy in mice as well.

Determination of Drug Release

The drug release characteristics of the two selected SEDDS formulation were comparatively evaluated with MZ powder and MZ solution in PEG 400. All four formulations were filled manually with a syringe into a hydrophilic airfill soft gelatin capsule (R. P. Scherer, Basking Ridge, N.J.) and the resulting hole was sealed thermally. USP XXII, Dissolution Apparatus 2 (VanKel Industrial, Inc.) was employed to characterize the release kinetics of MZ in vitro. Soft gelatin capsules containing the SEDDS, MZ solution, or MZ powder were individually placed in copper coils to keep the capsules at the bottom of their respective dissolution vessels. The vessels were filled with 500 mL of deionized water containing 0.5% Cremphor RH 40 (CMC=0.039%) at 37° C. Cremphor RH 40 was used to maintain the sink condition of the releasing medium. The release medium was constantly stirred at 50 rpm by a Teflon-coated dissolution paddle. Serial release samples were withdrawn and filtered through a 0.45 μm Millipore filter, and analyzed by the HPLC assay.

Studies of Cell Growth Inhibition in NSCLC and Skin Cancer Cells Using Formulation E4 and Placebo

Cells were plated in 96-well plates at 5×10⁶ cells/ml of Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal serum albumin, and incubated at 37° C. in a 5% CO₂/95% air-humidified incubator for 24 hrs. Cells were incubated for 48 hours after a medium change with fresh DMEM containing serial dilutions of E4 formulation or placebo. Cell survival was monitored with MTT assay. Anti-tumor effects of MZ were also evaluated using cell viability assays (trypan blue counting).

Example 2

Preclinical Bioavailability of Microemulsion E4

Animals

Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Ind.), weighing 300-400 g, were used after a 7-day acclimation period. Animals were housed under a 12 h light/dark cycle. A permanent catheter was implanted in the right jugular vein of each rat on the day before the study, under the anesthesia with ketamine:acetopromazine:xylazine (50:3.3:3.3 mg/kg). Animals were fasted overnight, while water was allowed ad libitum. On the morning of the experiment, rats were placed in individual metabolism cages.

Dosage Forms

The rats were randomized into three groups of three or four each. Rats in Group 1 were injected intravenously through the jugular catheter with a single dose (3.25 mg/kg) of the P7 formulation in a cosolvent system. Group 2 received oral microemulsion formulation E4; at a single dose of 5 mg/kg by oral gavage using an animal feeding needle (18×3″W/2¼ mm Ball). Group 3 were dosed with MZ aqueous unformulated suspension at 50 mg/kg similarly by oral gavage. The MZ suspension was prepared at 10 mg/ml with a trace amount of glycerin as a wetting agent.

Blood Sampling

Serial blood samples approximately 0.6 ml each were collected in heparinized tube through the catheter at determined time intervals for up to 24 hours. The blood samples were immediately centrifuged at 14,000 rpm for 3 min and the supernatant, plasma, was transferred into a 1.7-ml microcentrifuge tube and immediately stored at −80° C. until the HPLC assay.

HPLC Assay of Plasma Samples

MZ concentrations in plasma samples were determined by the HPLC method described in Example 1 with the following modifications. Briefly, 300 μl of plasma were mixed with 300 μl of acetonitrile to precipitate proteins. The acetonitrile contained 2 μg/mL quinidine (QD) as internal standard. Prednisone was not used as IS for plasma samples, because it was interfered by the broad solvent front from plasma blank. The mixture of plasma and acetonitrile was vortexed and centrifuged, then 100 μl of the supernatant were withdrawn and injected directly onto the HPLC system. The mobile phase consisted of 35% (v/v) acetonitrile in 0.05 M aqueous solution of KH₂PO₄, pH 6.5. The flow rate was 1.2 ml/min, and the effluent was monitored at 313 nm (0.01 AUFS).

Pharmacokinetic Analysis

Pharmacokinetic parameters were determined by fitting the plasma concentration-time data for each rat by a WinNonlin program. The maximum (peak) concentrations and the time to achieve maximum concentrations (peak time) in plasma were determined from the computer fitted plot. Areas under the concentration vs. time profiles from 0 to the last time point (720 min) of the measured concentration (AUC_(0→720)) was determined by the linear trapezoidal rule, and the AUC from the time of the last measured concentration to infinity (AUC_(720→∞)) was determined by dividing the last determined concentration by the terminal phase elimination rate constant, which was initially estimated from the least squares slope of the terminal linear segment of the plot. The terminal phase half-life was calculated as 0.693 divided by the terminal phase elimination rate constant. Other non-compartmental pharmacokinetic parameters were generated by area/moment analysis. Total plasma clearance (CL_(T)) was calculated from Dose/AUC, mean residence time (MRT) was calculated from AUMC/AUC, and steady-state volume of distribution (V_(ss)) was calculated from [(Dose AUMC)/AUC²]. For oral administration, bioavailability (F) was determined by the following equation: F=[AUC _(oral) /AUC _(iv)]×[Dose_(iv)/Dose_(oral)]×100% Statistical Analysis

Differences between any two mean values were statistically evaluated using paired or unpaired Student's t-test subsequent to statistical verification that the associated variances were homogeneous or equal.

Example 3 HPLC Assay of MZ

The retention times of MZ and the IS (prednisone) were 9.3 min and 5.6 min, respectively, under the HPLC eluting conditions aforementioned in Example 1. In particular, the stationary phase was a C₁₈ column, 300×3.9 mm (Waters Corp.), the mobile phase was acetonitrile/0.05 M KH₂PO₄ (40:60 V.V; pH=6.5), the flow rate was 1.2 ml/min, the detection was UV at 254 nm, for integration a Milton Ray CI-4100 integrator was used. An authentic HPLC chromatogram of MZ is shown in FIG. 1. Standard curves of MZ were established in the concentration range of 0.05 μg/ml to 5.0 μg/ml.

For the assay modified for plasma samples, the retention time for MZ and the IS (QD) were 10 and 7 min, respectively. On each day of plasma sample assay, a calibration curve was constructed to calculate the MZ concentrations in plasma samples. The linearity of the curve was established in a MZ concentration range of 0.05 to 2.5 μg/ml, with r²≧0.998. The calibration curve was plotted with peak area ratio of MZ/QD versus the known MZ concentrations spiked in rat plasma blanks.

Example 4 MZ Solubility in Various Solvent Media

The solubility of MZ in water and in the fourteen (14) tested solvents are compiled in Table I.

TABLE I Solubility of MZ in Water and Fourteen Solvent Media at 25° C. Solvent Medium Solubility (μg/mL) Enhancement Factor Water 0.025 1 Methanol 55 2,200 PEG 400 528 21,120 Propylene glycol 196 7,840 Triacetin 34.8 1,392 DMA 4,000 160,000 DMSO 5,000 200,000 Super refined cottonseed oil 0.25 10 Olive oil 19.7 788 Super refined peanut oil 0.25 10 Super refined soybean oil 5.8 232 Captex 200 13.3 532 Captex 355 1.2 48 Miglyol 812 40.6 1,624 Myvacet 9-45 39.3 1,572

MZ is poorly soluble in water with the solubility of 0.025 μg/mL. The MZ solubility in other solvent media, except super refined cottonseed oil, super refined peanut oil, and Captex 355, were all significantly higher than its aqueous solubility, with enhancement factors ranging from 232 to 200,000 in the reference to MZ aqueous solubility.

The MZ was favorably soluble in the six pharmaceutical solvents (methanol, PEG 400, propylene glycol, triacetin, DMA, and DMSO) with enhancement factors of 1,392 to 200,000. PEG 400, DMA, and DMSO yielded the highest three solubilities among the six tested solvents. PEG 400 alone yielded a solubility of 0.528 mg/ml, still much lower than the target of 1.5 mg/mL.

DMA and DMSO freely dissolved MZ, but were not pharmaceutically acceptable to use as straight solvents. The LD₅₀ of these solvents in rodents are 3.1 gm/kg. intravenously and 7.92 gm/kg orally. The oral and parenteral pharmaceutical products so far approved by FDA contain no more than 40% of DMA or DMSO. Therefore, PEG 400, DMA, and DMSO were selected to formulate MZ parenteral co-solvent systems with various compositions and ratios.

The MZ solubilities in the remaining five oils (olive oil, super refined soybean oil, Captex 200, Miglyol 812, and Myvacet 9-45) were significantly higher than its aqueous solubility, with enhancement factors ranging from 232 to 1,624 (Table I). However, none of them alone could reach the target concentration of 1.5 mg/ml. All the five oils could be used to formulate MZ microemulsions.

Example 5 Development of Parenteral Formulations

Ten co-solvent systems, coded as P1-P10, containing PEG 400 and DMA or PEG 400 and DMSO with various ratios, were systematically evaluated for their capacity to dissolve MZ. The MZ solubility in individual co-solvent systems are tabulated in Table II.

TABLE II Co-solvent Formulations (P1-P10) of MZ for Parenteral Delivery Composition (Ratio) MZ Solubility Code PEG 400 DMA DMSO Water (mg/mL) Comment P1 2 2 0 0 0.574 Patent formula for Busulfan (U.S. Pat. No. 5,430,057; 5,559,148 and Australia Patent 694141) P2 2 2 0 1 0.459 Containing 40% of DMA P3 3 1 0 0 2.284 Optimal I.V. formulation containing DMA P4 3 1 0 1 1.827 Containing 20% of DMA P5 7 1 0 0 0.934 P6 7 1 0 2 0.747 Containing 10% of DMA P7* 7 0 1 0 1.831 Optimal I.V. formulation containing DMSO P8 7 0 1 2 1.440 Containing 10% of DMSO P9 3 0 1 0 3.488 P10 3 0 1 1 2.790 Containing 20% of DMSO *The selected parenteral formulation, P7

The developed parenteral formulations yielded MZ solubility in the range of 0.46-3.49 mg/ml, 18,360-139,520 times higher than its aqueous solubility. All the formulations except P2 yielded solubilities higher than that in straight PEG 400, in the range of 1.09-6.61 times the MZ solubility in PEG 400. Six among the ten formulations, P3, P4, P7, P8, P9 and P10, yielded MZ solubility higher than 1.44 mg/ml, and were considered promising and suitable for further in vitro and in vivo evaluations. Formulation P7 with MZ solubility of 1.83 mg/ml was selected for future in vivo bioavailability study, based on its lowest content of DMSO and thus the maximal safety among the six promising systems.

Example 6 Development of Microemulsion Formulations

Thirty-six microemulsions (A1-A6, B1-B-6, C1-C-6, D1-D6, E1-E6, and F1-F6) were formulated and their capacities to incorporate water and to dissolve MZ were evaluated. These systems contained an oil (super refined soybean oil, Captex 200, Miglyol 812, or Myvacet 9-45), a solvent (DMA or DMSO), a blend of a high HLB Surfactant Tween 80 and a low HLB Surfactant Arlacel 80 (in a ratio of 1:1 or 2:1), and an aqueous phase. Their compositions are given in Table III-A-III-F. In these Tables, the following codes are used: J=super refined soybean oil; K=Captex 200; M=Miglyol 812; N=Myvacet 9-45; D=DMA; E=DMSO; O=Tween 80; P=Arlacel 80.

Among the formulated microemulsions, twenty-two formulations (A1-A3, B1-B3, C1-C4, D1-D4, E1-E4, and F1-F4) yielded MZ concentrations in the range of 1.5-2.64 mg/ml, meeting the target concentration of 1.5 mg/ml. When the maximum water that could be incorporated was taken into consideration, sixteen of the twenty-two microemulsion formulations, A3, B3, C1-C4, D3-D4, E1-E4, and F1-F4, that incorporated 10% (by weight) or higher of aqueous medium, were considered more stable than the rest formulations. Twelve microemulsions, C1-C4, E1-E4 and F1-F4 were the most promising formulations. Microemulsion E4 was selected for in vitro drug release and future in vivo studies of bioavailability and anti-neoplastic activity. In release study, formulation A4 was also comparatively evaluated. A representative pseudo-ternary phase diagram indicating the compositions of microemulsions E1-E6 is shown in FIG. 2.

TABLE III-A Self-Emulsified Microemulsion Formulations (A1-A6) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) A1 — 50.6 — — 24 — 12.7 12.7 4 9.65 2.19 A2 — 44.9 — — 21.3 — 16.9 16.9 4.5 9.65 1.93 A3 — 40.4 — — 19.2 — 20.2 20.2 11 9.65 1.89 A4 — 28.9 — — 13.7 — 28.8 28.8 13 9.65 1.13 A5 — 25.3 — — 11.9 — 31.4 31.4 13 9.65 0.98 A6 — 13.4 — — 6.4 — 40.1 40.1 14 9.65 0.5

TABLE III-B Self-Emulsified Microemulsion Formulations (B1-B6) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) B1 — 47.9 — — 22.8 — 16 8 5.3 11.4 2.25 B2 — 41.9 — — 19.9 — 20.9 10.6 6.7 11.4 2 B3 — 36.2 — — 17.2 — 24.1 12.1 10.3 11.4 1.8 B4 — 24.8 — — 11.8 — 33.1 16.5 13.9 11.4 1.29 B5 — 24.4 — — 11 — 32.5 16.3 15.9 11.4 1.3 B6 — 11.1 — — 5 — 46.5 23.3 14.1 11.4 0.58

TABLE III-C Self-Emulsified Microemulsion Formulations (C1-C10) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) C1 — 50.1 — — — 12.6 12.6 12.6 12.1 9.65 2.64 C2 — 42.6 — — — 10.7 16.1 16.1 14.5 9.65 2.31 C3 — 38.4 — — — 9.6 19.2 19.2 13.6 9.65 2.06 C4 — 34.7 — — — 8.7 21.7 21.7 13.2 9.65 1.85 C5 — 26.5 — — — 6.7 26.7 26.7 13.4 9.65 1.42 C6 — 17.2 — — — 4.3 32.5 32.5 13.5 9.65 0.92

TABLE III-D Self-Emulsified Microemulsion Formulations (D1-D10) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) D1 — — — 50.6 24 — 12.7 12.7 0 9.65 2.25 D2 — — — 44.9 21.3 — 16.9 16.9 0 9.65 2 D3 — — — 36.4 17.3 — 18.2 18.2 9.9 9.65 1.8 D4 — — — 33.1 15.6 — 20.7 20.7 9.9 9.65 1.64 D5 — — — 25.5 12.1 — 25.5 25.5 11.4 9.65 1.29 D6 — — — 16.9 8.1 — 31.4 31.4 12.2 9.65 0.87

TABLE III-E Self-Emulsified Microemulsion Formulations (E1-E6) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) E1 56.5 — — — — 14.1 14.1 14.1 1.2 9.65 2.14 E2 44.2 — — — — 11.1 16.6 16.6 11.5 9.65 1.88 E3 40 — — — — 10 20 20 10 9.65 1.87 E4 35.7 — — — — 8.9 22.3 22.3 10.8 9.65 1.8 E5 27.4 — — — — 6.9 27.6 27.6 10.5 9.65 1.15 E6 19 — — — — 4.7 33.2 33.2 9.9 9.65 0.79 Note: *The selected Formulation E4 comprised of Oil J: Solvent E: Surfactant O: Surfactant P = 2 g: 0.5 mL: 1.25 g: 1.25 g

TABLE III-F Self-Emulsified Microemulsion Formulations (F1-F6) of MZ Composition (% by Weight) MZ Formulation Oil Solvent Surfactant H₂O Concentration Code J K M N D E O P Incorporation HLB (mg/mL) F1 — — 50.4 — — 12.6 12.7 12.7 11.6 9.65 2.14 F2 — — 42.4 — — 10.6 15.9 15.9 15.2 9.65 1.88 F3 — — 39.2 — — 9.8 19.6 19.6 11.8 9.65 1.67 F4 — — 34.7 — — 8.7 21.8 21.8 13 9.65 1.5 F5 — — 27.2 — — 6.8 27.3 27.3 11.4 9.65 1.15 F6 — — 18.4 — — 4.6 32.8 32.8 11.4 9.65 0.78

Example 7 MZ Release from Microemulsions A4 and E4

The comparative in vitro release kinetics of MZ from drug powder, solution in PEG 400, microemulsion A4 (containing DMA), and microemulsion E4 (the selected oral formulation, containing DMSO), respectively, were evaluated and summarized in Table IV. The cumulative release profiles are presented in FIG. 3. MZ was readily released from the microemulsions A4 and E4, with 78.7% and 74.2%, respectively, dissolved in 30 minutes, greatly enhanced as compared with that of unformulated drug powder, 0.3% in 60 minutes. The MZ solution in PEG 400 was precipitated when in contact with the release medium after the capsule shell was disintegrated. The MZ dissolved slowly thereafter, and reached 17.1% dissolved in 60 minutes (Table IV).

TABLE IV In Vitro Release of MZ (n = 3 independent runs) Cumulative MZ Released (% Dose) Time MZ powder MZ in PEG 400 Formulation A4* Formulation E4** (min) Mean (SD) Mean (SD) Mean (SD) Mean (SD) 10 0.01 0.86 3.9 0.48 0.012 1.1 60 26.7 0.083 0.04 (0.04) 2.7  1.55 (1.00) 59.8  41.23 (32.33) 43.9  23.69 (21.87) 20 0.035 9.0 76.8 72.2 0.22 9.7 67 61.3 0.18 0.15 (0.10) 9.9  9.53 (0.47) 78.8 74.20 (6.32) 71.1 68.20 (6.00) 30 0.14 20.9 74.9 67.6 0.25 10.2 83.4 74.3 0.28 0.22 (0.07) — 15.56 (7.57) 77.7 78.67 (4.33) 80.8 74.23 (6.60) 40 0.22 24.3 82.3 71.0 0.23 12.2 78 72.5 0.41 0.29 (0.11) 12.1 16.20 (7.02) 80.4 80.23 (2.16) 69.3 70.93 (1.60) 50 0.2 22.7 — 85.1 0.31 10.4 80 73.9 0.35 0.29 (0.08) 8.3 13.80 (7.78) 89.7 84.85 (6.86) 81.3 80.10 (5.70) 60 0.27 27.5 98.1 75.4 0.27 11.3 77 79.4 0.32 0.29 (0.03) 12.5 17.10 (9.03) 86.4  87.17 (10.57) 78.0 77.60 (2.03) *A4 Formulation - Oil K: Solvent D: Surfactant O: Surfactant P = 2 g: 1 mL: 2 g: 2 g **E4 Formulation - Oil J: Solvent E: Surfactant O: Surfactant P = 2 g: 0.5 mL: 1.25 g: 1.25 g

Example 8 Preclinical Bioavailability Evaluation of E4

Eleven rats were used for the preclinical bioavailability evaluations of the lead oral microemulsion formulation in three independent runs. The rats were randomly grouped into three treatment groups: (a) I.V. dose of 3.25 mg/kg, n=3, (b) oral E4 microemulsion dose of 4.89 mg/kg, n=4, and (c) oral suspension dose of 50 mg/kg, n=4 (Table V). Table V shows the pharmacokinetic parameters of MZ from P7, E4 and oral suspension formulations in rats.

TABLE V Pharmacokinetic Parameters of MZ from P7 (Cosolvent) Formulation, E4 (Microemulsion) and Oral Suspension Formulations in Rats Route Rat of Bioavail- Num- admini- Weight Dose Cmax AUC₀₋₇₂₀ Tmax t_(1/2) ability ber stration Dosage Form (g) (mg/kg) (ng/mL) (ng min/mL) (min) (min) (%) M IV Co-solvent 300 3.25 10849 733042 — 104 N IV Co-solvent 320 3.25 13747 876594 — 77 O IV Co-solvent 320 3.25 7699 914010 — 120 Mean ± SD    313 ± 12  3.25 10765 ± 3025 841635 ± 95766 100 ± 22 A Oral Microemulsion 325 4.89 867 526350 240 — 41.6 E Oral Microemulsion 365 4.89 794 327660 240 127 25.9 F Oral Microemulsion 375 4.89 697 337680 240 231 26.7 G Oral Microemulsion 360 4.89 1064 354270 120 129 28.0 Mean ± SD    356 ± 22  4.89  856 ± 155  386490 ± 93883 210 ± 60 162 ± 59 30.6 ± 7.4  C Oral Suspension 303 50 154 42165 300 115 0.33 H Oral Suspension 345 50 138 38970 360 102 0.30 I Oral Suspension 370 50 168 64140 360 191 0.50 J Oral Suspension 380 50 63 14940 240 — 0.12 Mean ± SD    350 ± 34  50  131 ± 47   40054 ± 20137 315 ± 57 136 ± 48 0.31 ± 0.16

Table VI shows the plasma concentrations (ng/ml) that were obtained in the three groups of treated rats at various time intervals following administration of the formulations.

TABLE VI Plasma Concentrations of Bioavailability Evaluation of E4 and Unformulated Suspension in Rats Plasma Concentration (ng/ml) IV E4 Suspension Time Rats (min) M N O A E F G C H I J 0 10849 13747 7700 0 0 0 0 0 0 0 0 5 13365 7191 7 9162 15 7553 12633 6272 30 4599 6527 4601 646 502 216 534 60 2539 2846 3430 562 490 310 854 22 26 35 75 14 90 1848 2371 2664 120 1654 1924 2235 842 555 533 1064 26 13 58 52 180 1078 1362 1587 71 240 781 842 1274 867 794 697 840 64 90 63 300 469 371 789 154 360 187 779 567 558 449 138 168 10 480 787 374 550 232 43 60 116 720 556 83 164 12 12 46

The plasma-time profiles of MZ from I.V., E4 and unformulated suspensions were constructed. (FIG. 4-6). The semi-log plot of the profiles of MZ after I.V. administration to individual rats (FIG. 7) were all bi-phasic with a rapid decline in mean concentrations from 7699 ng/ml to 2662 ng/ml within 90 min. The profile fitted a two-compartment model, indicating that the distribution of MZ in rats had a distinct tissue peripheral compartment. The distribution process appeared to be faster than the absorption process when MZ was given orally. The profiles of MZ from administrations of oral E4 and suspension did not exhibit any bi-phasic decline and fitted well into the typical 1-compartment model with an absorption process of first-order kinetics. The drug concentrations from suspension dosing (10-168 ng/ml) were substantial lower than those from E4 dosing (83-1064 ng/ml), even the suspension dose administered was 10 times higher.

The plasma profiles were analyzed by non-compartmental model with WinNonlin program to derive pharmacokinetic parameters for comparison. The MZ peak concentration from E4 was 856±155 ng/ml, 6.5 folds of that from suspension, 131±47 ng/ml. (Table V). The peak time from both dosing were comparable, 210±60 and 315±57 min, for E4 and suspension, respectively. The absorption half-lives were similar, 100±57 and 184±63 min, for E4 and suspension, respectively.

The biological half-life was not statistically different from the different formulations (I.V. solution, E4 microemulsion and suspension) nor by the various routes of administration (I.V. and oral.), ranging from 100-162 min.

The systemic exposure of MZ after the different dosage forms were measured by AUC₀₋₇₂₀. When the values were normalized by the respective doses given, the absolute bioavailability of E4 and suspension in reference to I.V. dosing were 30.6% and 0.31%, respectively (Table V). The relative bioavailability of E4 in reference to the unformulated suspension was 98.7. It means that by formulating MZ into the microemulsion formulation, the bioequivalent dose of E4 was about 1/100 of that of the unformulated suspension.

Example 9 Studies of Cell Growth Inhibition in NSCLC and Skin Cancer Cell Lines Using Formulation E4 and Placebo

The E4 formulation and placebo were used in studies of cell growth inhibition in skin cancer cell lines (SRB1, A375 and 2237) and non-small cell lung cancer (NSCLC) cell lines. The dose-response curves for cell growth inhibition were established for the range of 1.7-170 ng/ml for skin cancer cells (FIG. 8) and 2.5-250 ng/ml for NSCLC (FIG. 9). The IC₅₀ of MZ in skin cancer cells were 91, 129, and 171 ng/ml for SRB1, A375, and 2237, respectively (FIG. 8). The IC₅₀ of MZ from two microemulsion formulations in NSCLC cells were 8 and 10 ng/ml, respectively, which represented 8-9 fold enhancements, as compared with those of DMSO-dissolved MZ controls (FIG. 9). Thus, this study of enhanced anti-neoplastic activity in cultured cells provides evidence of the effective delivery of MZ by the E4 formulation.

Thus, the developed microemulsion formulations exhibited favorable characteristics in drug release, preclinical efficacy in cultures, and preclinical pharmacokinetics in rats.

All of the compositions disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are chemically, physiologically, and pharmacologically-related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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1. A pharmaceutical composition, comprising: a) a benzimidazole derivative having the formula:

wherein R³ is selected from the group consisting of H, carboxyl (—CO₂H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, 5-methoxy, fluorophenylmethyl-2-chloro, propenyl, chloropropyl or esters (—CO₂R⁴) wherein R⁴ is selected from the group consisting of alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH₃CH₂(OCH₂CH₂)n-, or CH₃CH₂CH₂(OCH₂CH₂CH₂)n-, or (CH₃)₂CH(OCH(CH₃)CH₂)n-, wherein n is from 1-3 wherein R¹ is OH, Cl, SH, carbamate or piperidin-4-yl, and R² is hydrogen, α-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof; b) PEG 400; c) a dipolar aprotic solvent; and d) water, wherein the ratio of PEG 400:dipolar aprotic solvent:water by weight is 3:1:1 or 7:1:2.
 2. The pharmaceutical composition of claim 1, wherein the dipolar aprotic solvent is N,N-dimethylacetamide.
 3. The pharmaceutical composition of claim 1, wherein the dipolar aprotic solvent is dimethylsulfoxide.
 4. A pharmaceutical composition, comprising: a) a benzimidazole derivative having the formula:

wherein R³ is selected from the group consisting of H, carboxyl (—CO₂H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, 5-methoxy, fluorophenylmethyl-2-chloro, propenyl, chloropropyl or esters (—CO₂R⁴) wherein R⁴ is selected from the group consisting of alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH₃CH₂(OCH₂CH₂)n-, or CH₃CH₂CH₂(OCH₂CH₂CH₂)n-, or (CH₃)₂CH(OCH(CH₃)CH₂)n-, wherein n is from 1-3 wherein R¹ is OH, Cl, SH, carbamate or piperidin-4-yl, and R² is hydrogen, α-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof; b) a surfactant; c) a mixture of an oil and a dipolar aprotic solvent; wherein the ratio of oil to dipolar aprotic solvent is about 2:1 by weight; and d) water.
 5. The pharmaceutical composition of claim 4, wherein the benzimidazole derivative is methyl 5-benzoylbenzimidazole-2-carbamate.
 6. The pharmaceutical composition of claim 4, wherein the benzimidazole derivative is methyl 5-(phenylthio)-2-carbamate.
 7. The pharmaceutical composition of claim 4, wherein the benzimidazole derivative is


8. The pharmaceutical composition of claim 4, wherein the benzimidazole derivative is


9. The pharmaceutical composition of claim 4, wherein the benzimidazole derivative is


10. The pharmaceutical composition of claim 4, wherein the oil is super refined corn oil, super refined cottonseed oil, olive oil, super refined peanut oil, super refined soybean oil, Captex 200, Captex 355, Miglyol 812, or Myvacet 9-45.
 11. The pharmaceutical composition of claim 10, further comprising one or more additional oils.
 12. The pharmaceutical composition of claim 4, wherein the oil is Captex
 200. 13. The pharmaceutical composition of claim 4, wherein the oil is Miglyol
 812. 14. The pharmaceutical composition of claim 4, wherein the surfactant is Tween
 80. 15. The pharmaceutical composition of claim 4, wherein the surfactant is Arlacel
 80. 16. The pharmaceutical composition of claim 4, further defined as comprising more than one surfactant.
 17. The pharmaceutical composition of claim 16, wherein the surfactants include Tween 80 and Arlacel
 80. 18. The pharmaceutical composition of claim 4, further defined as a microemulsion.
 19. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is suitable for oral delivery to a subject.
 20. A pharmaceutical composition of, comprising: (a) methyl 5-benzoylbenzimidazole-2-carbamate; (b) 10%-60% by weight of an oil; (c) 2% -30% by weight of a dipolar aprotic solvent; (d) 5% -50% by weight of a first surfactant; and (e) 5% -50% by weight of a second surfactant, wherein the ratio of oil to dipolar aprotic solvent is about 2:1 to 4:1 by weight.
 21. The pharmaceutical composition of claim 20, wherein the first surfactant is Tween 80 and the second surfactant is Arlacel
 80. 22. The pharmaceutical composition of claim 21, comprising 13% to 90% by weight of a Captex 200 and N,N dimethylacetamide mixture, 5%-50% by weight of Tween 80, 5%-50% by weight of Arlacel 80, and 2%-20% by weight of water.
 23. The pharmaceutical composition of claim 22, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is 9.3 mg/ml-3.0 mg/ml.
 24. The pharmaceutical composition of claim 22, comprising 28.9% by weight of a Captex 200, 13.7% by weight of N,N dimethylacetamide, 28.8% by weight of Tween 80, 28.8% by weight of Arlacel 80, and 13% water.
 25. The pharmaceutical composition of claim 24, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 12 mg/ml.
 26. The pharmaceutical composition of claim 21, comprising 10%-60% by weight of a Captex 200, 2%-20% by weight of dimethylsulfoxide, 10%-40% by weight of Tween 80, 10%-40% by weight of Arlacel 80, and 10%-20% by weight of water.
 27. The pharmaceutical composition of claim 26, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 0.5 mg/ml-3.0 mg/ml.
 28. The pharmaceutical composition of claim 26, comprising 50.1% by weight of a Captex 200, 12.6% by weight of dimethylsulfoxide, 12.6% by weight of Tween 80, 12.6% by weight of Arlacel 80, and 12.1% by weight of water.
 29. The pharmaceutical composition of claim 28, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 2.6 mg/ml.
 30. The pharmaceutical composition of claim 26, comprising 42.6% by weight of a Captex 200, 10.7% by weight of dimethylsulfoxide, 16.1% by weight of Tween 80, 16.1% by weight of Arlacel 80, and 14.5% by weight of water.
 31. The pharmaceutical composition of claim 30, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 23 mg/ml.
 32. The pharmaceutical composition of claim 26, comprising 38.4% by weight of a Captex 200, 9.6% by weight of dimethylsulfoxide, 19.2% by weight of Tween 80, 19.2% by weight of Arlacel 80, and 13.6% by weight of water.
 33. The pharmaceutical composition of claim 32, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 2.0 mg/ml.
 34. The pharmaceutical composition of claim 26, comprising 34.7% by weight of a Captex 200, 8.7% by weight of dimethylsulfoxide, 21.7% by weight of Tween 80, 21.7% by weight of Arlacel 80, and 13.2% by weight of water.
 35. The pharmaceutical composition of claim 34, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.85 mg/ml.
 36. The pharmaceutical composition of claim 21, comprising 10%-60% by weight of super refined soybean oil, 2%-20% by weight of dimethylsulfoxide, 10%-40% Tween 80, 10%-40% Arlacel 80, and 0.5%-15% by weight of water.
 37. The pharmaceutical composition of claim 36, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is 0.5 mg/m-2.5 mg/ml.
 38. The pharmaceutical composition of claim 36, comprising 56.5% by weight of super refined soybean oil, 14.1% by weight of dimethylsulfoxide, 14.1% Tween 80, 14.1% Arlacel 80, and 12% by weight of water.
 39. The pharmaceutical composition of claim 38, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 2.1 mg/ml.
 40. The pharmaceutical composition of claim 36, comprising 44.2% by weight of super refined soybean oil, 11.1% by weight of dimethylsulfoxide, 16.6% Tween 80, 16.6% Arlacel 80, and 11.5% by weight of water.
 41. The pharmaceutical composition of claim 40, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.9 mg/ml.
 42. The pharmaceutical composition of claim 36, comprising 40.0% by weight of super refined soybean oil, 10.0% by weight of dimethylsulfoxide, 20.0% Tween 80, 20.0% Arlacel 80, and 10.0% by weight of water.
 43. The pharmaceutical composition of claim 42, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.9 mg/ml.
 44. The pharmaceutical composition of claim 36, comprising 35.7% by weight of super refined soybean oil, 89% by weight of dimethylsulfoxide, 22.3% Tween 80, 22.3% Arlacel 80, and 10.8% by weight of water.
 45. The pharmaceutical composition of claim 44, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.8 mg/ml.
 46. The pharmaceutical composition of claim 21, comprising 50.4% by weight of Myglyol 812, 12.6% by weight of dimethylsulfoxide, 12.7% by weight of Tween 80, 12.7% by weight of Arlacel 80, and 11.6% by weight of water.
 47. The pharmaceutical composition of claim 46, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 2.1 mg/ml.
 48. The pharmaceutical composition of claim 21, comprising 42.4% by weight of Myglyol 812, 10.6% by weight of dimethylsulfoxide, 15.9% by weight of Tween 80, 15.9% by weight of Arlacel 80, and 15.2% by weight of water.
 49. The pharmaceutical composition of claim 48, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.9 mg/ml.
 50. The pharmaceutical composition of claim 21, comprising 32.9% by weight of Myglyol 812, 9.8% by weight of dimethylsulfoxide, 19.6% by weight of Tween 80, 19.6% by weight of Arlacel 80, and 11.8% by weight of water.
 51. The pharmaceutical composition of claim 50, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.7 mg/ml.
 52. The pharmaceutical composition of claim 21, comprising 34.7% by weight of Myglyol 812, 8.7% by weight of dimethylsulfoxide, 21.8% by weight of Tween 80, 21.8% by weight of Arlacel 80, and 13.0% by weight of water.
 53. The pharmaceutical composition of claim 52, wherein the methyl 5-benzoylbenzimidazole-2-carbamate concentration is about 1.5 mg/ml. 